Oligonucleoside methylphosphonates have been used to study the function of specific RNA sequences in biochemical and intact cellular systems. See Miller et al, Biochemistry 20, 1874-1880 (1981) and Jayaraman et al, Proc. Nat'l Acad. Sci. 78, 1537-1541 (1981). Since these nonionic nucleic acid analogs can be taken up intact by mammalian cells and certain bacterial cells in culture, these compounds promise to be useful reagents for exploring and regulating the function of nucleic acids within living cells. See, in this regard, the above-mentioned U.S. application Ser. No. 363,230 and U.S. Pat. No. 4,469,863.
Various procedures have previously been described for synthesizing oligonucleoside methylphosphonates and the like. Thus, the synthesis of oligonucleoside methylphosphonates on a silica gel support has previously been described (Miller et al (1983) Nucleic Acids Res., 11, 5189-5204). In that work, protected nucleoside 3'-methyl-phosphonic chlorides or tetrazolides were used as synthetic intermediates. While oligothymidine methylphosphonates could be efficiently synthesized by this procedure, low yields were encountered when other nucleosides, particularly d-[(MeO).sub.2 Tr]ibuG, were used.
The preparation of oligonucleoside methylphosphonates on a glass support using nucleoside 3'-methylphosphine chlorides as reactive intermediates has also been described (Sinha et al, Tetrahedron Letters, 24, 877-880, 1983).
In U.S. Pat. No. 4,507,433, we have described an improved method for synthesizing oligodeoxyribonucleoside alkyl or arylphosphonates, notably methylphosphonates, of preselected sequence and length in high yield which comprises condensing (1) a 5'-protected nucleoside 3'-alkyl or arylphosphonate alkyl-ammonium salt with (2) a nucleoside or oligomer thereof bound to a solid or insoluble polymer support, e.g. a crosslinked polystyrene support. The condensation can be repeated as many times as necessary, with the same nucleoside (1) or a different one, and using the previously obtained oligomer product after deprotecting the 5-position as reactant (2), to ultimately obtain an oligonucleoside phosphonate having the desired sequence and length. Typically the invention may be used to prepare an oligomer containing up to nine nucleoside units or even more.
The invention of U.S. Pat. No. 4,507,433 also includes convenient means, notably the use of a diamino alkane, preferably ethylene diamine dissolved in ethanol, for cleaving the oligomer product from the supporting polymer.